Project Summary Mitochondria are important organelles within the cell that participate in a number of cellular processes including the generation of energy through oxidative phosphorylation. Mitochondria possess their own genome that is vital to mitochondrial function including oxidative phosphorylation. Mutations within this genome can cause mitochondrial dysfunction. A link has been established between obesity and mitochondrial dysfunction. It has recently been proposed that mutations to mitochondrial DNA might be one of the mechanisms that lead to whole body metabolic dysfunction. We have preliminary evidence that mitochondrial DNA mutations might induce mitochondrial dysfunction and impair whole body metabolic homeostasis thus ultimately leading to an obesity phenotype. Mitochondrial DNA is replicated and repaired by a single DNA polymerase (mitochondrial DNA polymerase gamma, or PolG). Work utilizing a homozygous whole body knock-in mutation within PolG (PolG-/-) results in a supraphysiological increase in both mitochondrial DNA point mutations and deletions and a progeroid like phenotype within mice. Although the accumulation of mtDNA mutations is associated with mitochondrial dysfunction in these mice, the drastically reduced life span of the mice make them a poor model to study the association of mtDNA mutations and metabolic function. We have collected preliminary data utilizing a heterozygous version of the same mouse model (PolG+/-). Our data show an obese phenotype and a more physiologically relevant level of mitochondrial DNA mutations that might precede mitochondrial dysfunction and further alter whole body metabolic homeostasis. Using the heterozygous PolG+/- mouse model, this proposal will seek to establish a link between mitochondrial DNA mutations, mitochondrial dysfunction, and whole body metabolic homeostasis. The first aim of the proposal will identify if mitochondrial DNA mutation load impacts mitochondrial function and dynamics and whole body metabolic homeostasis. The second aim of the proposal will examine if skeletal muscle overexpression of heat shock protein 72 (HSP72), a proposed positive regulator of mitochondrial function, can rescue the metabolic dysfunction via a reduction in mitochondrial DNA mutation load. To accomplish this, we have already generated both whole body heterozygous PolG mice (PolG+/-) and PolG+/- mice crossed with muscle specific HSP72 overexpression via a transgene (PolG+/- x HSP72mTg). Whole body and tissue specific metabolic homeostasis will be assessed along with factors that impact mitochondrial biogenesis, dynamics, and degradation. The proposed studies will fill a knowledge gap and address the question of a potential link between mitochondrial DNA mutations and metabolic dysfunction.